Natalia Guillén

Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver

Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote fatty liver in mice, while cis-9, trans-11-CLA ameliorates this effect, suggesting regulation of multiple genes. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid isomers, and their hepatic gene expression was analyzed with DNA microarrays. To provide an initial screening of candidate genes, only 12 with remarkably modified expression between both CLA isomers were considered and confirmed by quantitative RT-PCR. Additionally mRNA expression of 15 genes involved in lipid metabolism was also studied. Ten genes (Fsp27, Aqp4, Cd36, Ly6d, Scd1, Hsd3b5, Syt1, Cyp7b1, and Tff3) showed significant associations among their expressions and the degree of hepatic steatosis. Their involvement was also analyzed in other models of steatosis. In hyperhomocysteinemic mice lacking Cbs gene, only Fsp27, Cd36, Scd1, Syt1, and Hsd3b5 hepatic expressions were associated with steatosis. In apoE-deficient mice consuming olive-enriched diet displaying reduction of the fatty liver, only Fsp27 and Syt1 expressions were found associated. Using this strategy, we have shown that expression of these genes is highly associated with hepatic steatosis in a genetic disease such as Cbs deficiency and in two common situations such as Western diets containing CLA isomers or a Mediterranean-type diet. Conclusion: The results highlight new processes involved in lipid handling in liver and will help to understand the complex human pathology providing new proteins and new strategies to cope with hepatic steatosis.

Understanding the role of dietary components on atherosclerosis using genetic engineered mouse models.

The generation by genetic engineering of two murine models to investigate atherosclerosis, such as the apoE- and LDLr- deficient mice, is providing an extraordinaire knowledge of the effect of different nutrients on this complex disease. The present revision provides a comprehensive overview of the advances in this field that point to a remarkable complexity. While some controversies over puzzling results could be explained invoking potential nutrient interactions or different food sources of nutrients, it also appears that other factors such as sex, genetic background or immunological status are emerging as generators of differential responses to nutrients during the atherosclerotic process.

Cystathionine β-synthase deficiency causes infertility by impairing decidualization and gene expression networks in uterus implantation sites

Hyperhomocysteinemia has been reported in human reproduction as a risk factor for early pregnancy loss, preeclampsia, and congenital birth defects like spina bifida. Female infertility was also observed in cystathionine beta synthase-deficient mice (Cbs-KO) as an animal model for severe hyperhomocysteinemia. The aim for the present research was to elucidate the time-point of pregnancy loss and to pinpoint gene and cellular changes involved in the underlying pathological mechanism. By mating 90-day-old wild-type and Cbs-KO female mice with their homologous male partners, we found that pregnancy loss in Cbs-KO occurred between the 8th and 12th gestation day during placenta formation. DNA microarrays were carried out on uterus from implantation and interimplantation samples obtained on day 8. The results allowed us to select genes potentially involved in embryo death; these were individually confirmed by RT-qPCR, and their expressions were also followed throughout pregnancy. We found that changes in expression of Calb1, Ttr, Expi, Inmt, Spink3, Rpgrip1, Krt15, Mt-4, Gzmc, Gzmb, Tdo2, and Afp were important for pregnancy success, since a different regulation in Cbs-KO mice was found. Also, differences in relationships among selected genes were observed, indicating a dysregulation of these genes in Cbs-KO females. In conclusion, our data provide more information on the gene expression cascade and its timely regulated process required for a successful pregnancy. In addition, we unveil new potential avenues to explore further investigations in pregnancy loss.

Lipopolysaccharide Induces Inhibition of Galactose Intestinal Transport in Rabbits in vitro

Background/Aims: Previous studies from our laboratory have revealed impaired intestinal absorption of D-galactose in lipopolysaccharide-treated rabbits. The aim of the present work was to examine the effect of LPS on D-galactose intestinal absorption in vitro. Methods: D-galactose intestinal transport was assessed employing three techniques: sugar uptake in rings of everted jejunum, transepithelial flux in Ussing-type chambers and transport assays in brush border membrane vesicles. The level of expression of the Na+/D-galactose cotransporter (SGLT1) was analyzed by Western blot. Results: LPS decreased the mucosal D-galactose transport in rabbit jejunum but a preexposition to the endotoxin was required. LPS affected the Na+-dependent transport system by increasing the apparent Km value without affecting the Vmax. It also decreased the Na+, K+-ATPase activity. However, it did not inhibit neither the uptake of D-galactose by brush border membrane vesicles nor modified the SGLT1 protein levels in the brush border, suggesting an indirect endotoxin effect. This inhibitory effect, was reduced by selective inhibitors of Ca2+-calmodulin (W13), protein kinase C (GF 109203X), p38 mitogen-activated protein kinase (SB 203580), c-Jun N-terminal kinase (SP 600125) and mitogen extracellular kinase (U 0126). Conclusion: LPS inhibits the mucosal Na+-dependent D-galactose intestinal absorption and the Na+, K+-ATPase activity when it is added to the tissue. Intracellular processes related to protein kinases seem to be implicated in the endotoxin effect.

Proteomics and gene expression analyses of squalene-supplemented mice identify microsomal thioredoxin domain-containing protein 5 changes associated with hepatic steatosis.

Squalene is an abundant hydrocarbon present in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE-knock-out mice. To study its effects on microsomal proteins, 1g/kg/day of squalene was administered to those mice. After 10 weeks, hepatic fat content was assessed and protein extracts of microsomal enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by peptide fingerprinting and MSMS analysis. Squalene administration modified the expression of thirty-one proteins involved in different metabolic functions and increased the levels of those involved in vesicle transport, protein folding and redox status. Only mRNA levels of 9 genes (Arg1, Atp5b, Cat, Hyou1, Nipsnap1, Pcca, Pcx, Pyroxd2, and Txndc5) paralleled these findings. No such mRNA changes were observed in wild-type mice receiving squalene. Thioredoxin domain-containing protein 5 (TXNDC5) protein and mRNA levels were significantly associated with hepatic fat content in apoE-ko mice. These results suggest that squalene action may be executed through a complex regulation of microsomal proteins, both at the mRNA and post-transcriptional levels and the presence of apoE may change the outcome. Txndc5 reflects the anti-steatotic properties of squalene and the sensitivity to lipid accumulation.

Postprandial Changes in High Density Lipoproteins in Rats Subjected to Gavage Administration of Virgin Olive Oil

Background and Aims The present study was designed to verify the influence of acute fat loading on high density lipoprotein (HDL) composition, and the involvement of liver and different segments of small intestine in the changes observed. Methods and Results To address these issues, rats were administered a bolus of 5-ml of extra-virgin olive oil and sacrificed 4 and 8 hours after feeding. In these animals, lipoproteins were analyzed and gene expressions of apolipoprotein and HDL enzymes were assessed in duodenum, jejunum, ileum and liver. Using this experimental design, total plasma and HDL phospholipids increased at the 8-hour-time-point due to increased sphingomyelin content. An increase in apolipoprotein A4 was also observed mainly in lipid-poor HDL. Increased expression of intestinal Apoa1, Apoa4 and Sgms1 mRNA was accompanied by hepatic decreases in the first two genes in liver. Hepatic expression of Abcg1, Apoa1bp, Apoa2, Apoe, Ptlp, Pon1 and Scarb1 decreased significantly following fat gavage, while no changes were observed for Abca1, Lcat or Pla2g7. Significant associations were also noted for hepatic expression of apolipoproteins and Pon1. Manipulation of postprandial triglycerides using an inhibitor of microsomal transfer protein -CP-346086- or of lipoprotein lipase –tyloxapol- did not influence hepatic expression of Apoa1 or Apoa4 mRNA. Conclusion All these data indicate that dietary fat modifies the phospholipid composition of rat HDL, suggesting a mechanism of down-regulation of hepatic HDL when intestine is the main source of those particles and a coordinated regulation of hepatic components of these lipoproteins at the mRNA level, independently of plasma postprandial triglycerides.

Cysteinemia, rather than homocysteinemia, is associated with plasma apolipoprotein A-I levels in hyperhomocysteinemia: lipid metabolism in cystathionine β-synthase deficiency.

OBJECTIVE Genetic and dietary hyperhomocysteinemia has been found to decrease high density lipoproteins (HDL) and their apolipoprotein A1 (APOA1). To test the hypothesis that the presence of cysteine could normalize HDL levels in hyperhomocysteinemic cystathionine beta-synthase (Cbs)-deficient mice and that the inclusion of glycine would block this effect. METHODS Lipids and HDL cholesterol were studied in Cbs-deficient mice and wild-type animals fed a low-methionine diet supplemented with cysteine and glycine and in Cbs-deficient mice on the same diet supplemented only with cysteine. RESULTS Triglyceride and homocysteine levels were significantly decreased and increased, respectively in Cbs-deficient mice irrespective of treatment. However, plasma cholesterol, glucose and APOA1 were significantly decreased in homozygous Cbs-deficient mice when they received the cysteine and glycine-enriched beverage. This group of mice also showed decreased mRNA levels and increased hepatic content of APOA1 protein, the latter increase was observed in endothelial cells. A significant, inverse relationship was observed between plasma and hepatic APOA1 concentrations while a positive one was found between plasma levels of cysteine and APOA1. CONCLUSION These data suggest an altered hepatic management of APOA1 and that cysteine may be involved in the control of this apolipoprotein at this level. Overall these findings represent a new aspect of dietary regulation of HDL at the hepatic transendothelial transport.

Apolipoprotein E determines the hepatic transcriptional profile of dietary maslinic acid in mice.

The hypothesis that the maslinic acid (MA) of olive oil (OO) dramatically influences hepatic gene expression was tested in mice. Two OOs only differing in the presence of MA were prepared. Using DNA microarrays, we analyzed hepatic gene expression in apolipoprotein E (apoE)-deficient mice with a C57BL/6J genetic background that were fed with isocaloric, isonitrogenous diets containing either 10% (w/w) OO or 10% MA-enriched OO. As an initial screening of potential candidate genes involved in a differential response, this study further considered only genes with remarkably modified expression (signal log(2) ratio higher than1.5 or lower than -1.5). The nine genes fulfilling these prerequisites were confirmed by quantitative reverse transcriptase polymerase chain reaction and analyzed in C57BL/6J wild-type mice. Only Cyp2b9, Cyp2b13 and Dbp expressions appeared significantly increased, and Marco was significantly decreased in apoE-deficient mice receiving the MA-enriched diet. Dbp was up-regulated to an extent depending on the genetic background of the mice and negatively associated with the expression of Marco, a gene strongly up-regulated by the absence of apoE. These expression changes could be used as markers of the intake of the MA-enriched OO and are influenced by genetic background generated by the absence or the presence of apoE. Overall, these results (a) indicate that MA in virgin OO is highly active in controlling hepatic gene expression and (b) highlight the important interaction between the response to MA and the presence of apoE. They also confirm that virgin OO cannot be simplistically classified as monounsaturated fatty-enriched oil without paying attention to its active minor components.

Proteomics and gene expression analyses of mitochondria from squalene-treated apoE-deficient mice identify short-chain specific acyl-CoA dehydrogenase changes associated with fatty liver amelioration

Squalene, a hydrocarbon involved in cholesterol biosynthesis, is an abundant component in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE knock-out mice. To study the effect of squalene on mitochondrial proteins in fatty liver, 1 g/kg/day of this isoprenoid was administered to those mice. After 10 weeks, hepatic fat was assessed and protein extracts from mitochondria enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by MS analysis. Squalene administration modified the expression of eighteen proteins involved in different metabolic processes, 12 associated with hepatic fat content. Methionine adenosyltransferase I alpha (Mat1a) and short-chain specific acyl-CoA dehydrogenase (Acads) showed significant increased and decreased transcripts, respectively, consistent with their protein changes. These mRNAs were also studied in wild-type mice receiving squalene, where Mat1a was found increased and Acads decreased. However, this mRNA was significantly increased in the absence of apolipoprotein E. These results suggest that squalene action may be executed through a complex regulation of mitochondrial protein expression, including changes in Mat1a and Acads levels. Indeed, Mat1a is a target of squalene administration while Acads reflects the anti-steatotic properties of squalene.

Knowledge of the Biological Actions of Extra Virgin Olive Oil Gained From Mice Lacking Apolipoprotein E

The low incidence of cardiovascular disease in countries bordering the Mediterranean basin, where olive oil is the main source of dietary fat, has stimulated interest in the chemical composition of olive oil and in the production of other oils enriched with its minor components. This review summarizes what has been learned about the effects of different olive oil preparations on the development of atherosclerosis and about the prognostic value of associated plasma variables in the disease from experiments on genetically modified mice that spontaneously develop atherosclerosis. The limitations of this animal model associated with its morphological and physiological differences with humans are minimized by the similarity of the two genomes and by the potential for increased understanding attainable, given that the dietary interventions reported here would have taken 400 years to achieve in humans. As observed in traditional Mediterranean populations, it has been confirmed that extra virgin olive oil is beneficial when consumed judiciously and in a diet that is low in cholesterol due to the relative scarcity of animal products. Furthermore, the use of genomic techniques has led to the identification of new markers of response to olive oil. In conclusion, multidisciplinary research into extra virgin olive oil is expanding our knowledge of the substances biological properties.